Excessive oxidafive and nitrosative (O/N) activifies in the brain have been recognized as the basis for the neuronal excitotoxicity and glial cell inflammatory responses that underiie many neurodegenerative diseases, including stroke. Recent studies have discovered the role of NADPH oxidase as an important source of reactive oxygen species (ROS) in brain cells. Activafion of NADPH oxidase is associated with a number of downstream signaling pathways, including the NF-KB pathway that induces INOS and produces nitric oxide (NO). Among other reactions, NO can regulate protein structure and function by interacfing with protein reactive cysteine thiol residues to form S-nitrosothiol. Many proteins associated with NADPH oxidase signaling pathways appear to be modified by S-nitrosylation. While the mechanisms are unknown, there is considerable evidence to suggest that a number of botanical compounds may protect the brain against O/N insults, including those resulfing from cerebral ischemia. In keeping with the central goal ofthis Center, Project 2 will identify new botanicals and test the hypothesis that stroke-mediated neuronal excitotoxicity and glial inflammatory responses are due in part to ROS from NADPH oxidase and subsequent S-nitrosylafion of key proteins in the NADPH oxidase signaling pathways. In Aim 1. neurons and astrocytes/microglia will be used to screen botanical extracts and pure compounds isolated from the extracts for their ability to suppress NMDA-mediated neuronal excitotoxicity and cytokine-induced inflammatory responses in glial cells. The effects of botanicals on S-nitrosylation of proteins and differentially-expressed mRNA and proteins in neurons and glial cells will be invesfigated using the newly developed NitroDIGE protocol and mRNA megasequencing facilities in the Interactions Core. In Aim 2, the mouse focal cerebral ischemia model induced by occlusion of the middle cerebral artery will be used to investigate whether dietary supplementation with specific botanicals offers protective effects against cerebral ischemia/reperfusion damage. These studies will include using mice deficient in N0X2 and iNOS to test the effects of botanicals on behavioral outcome, neuronal survival, glial activation, and inflammatory responses. Aim 3 will test the hypothesis that identified botanicals offers neuroprotective effects by reducing cerebral ischemia/reperfusion damage through activation of the Nrf2/Keap1 antioxidant signaling pathway. In these studies, astrocyte- or neuron-targeted conditional Keapl-null mice will be used. In Aims 2 and 3, we will also identify changes in S-nitrosylated and inflammatory proteins and mRNA associated with oxidafive and anfi-oxidafive signaling pathways in disfinct brain regions. The in vitro and in vivo approaches of Project 2, together with support from the unique and state-of-the-art core facilities of this Center, will combine to identify specific cellular and molecular mechanisms through which novel botanicals and/or their active components may exert beneficial effects and protect against stroke damage.